Depigmenting dermatological and/or cosmetic composition

ABSTRACT

PCT No. PCT/FR96/00422 Sec. 371 Date Sep. 23, 1997 Sec. 102(e) Date Sep. 23, 1997 PCT Filed Mar. 21, 1996 PCT Pub. No. WO96/29050 PCT Pub. Date Sep. 26, 1996A dermatological and/or cosmetic composition containing a depigmenting active extract of mouse-ear hawkweed and the use thereof in a cosmetic treatment method are disclosed. The use of an active substance obtainable from mouse-ear hawkweed for preparing a depigmenting active medicament is also disclosed.

This application claims priority under 35 USC 371 based uponPCT/FR96/00422 filed Mar. 21, 1996 and under 35 USC 119 based upon FR 9503425 filed Mar. 23, 1995.

The present invention relates to novel dermocosmetic compositions whichhave depigmenting activity.

The color of the skin is due to several substances: haemoglobin in theblood vessels, carotenoids in the dermis and, especially, melanin in theepidermis.

This melanin is produced by melanocytes in the basal layer, under theaction of tyrosinase, copper and oxygen.

Under the effect of exogenous or endogenous stimulations, changes inskin color may appear. These are dyschromias.

A change in the skin pigmentation may take place:

either by excess: hyperchromia,

or by deficit: hypochromia.

This may take place in the epidermis or in the dermis and may be due toa variation in the amount of melanin or in the number of melanocytes.

Hyperchromias are accumulations of melanin pigments, carotenoids orexogenous pigments.

The melanin in the skin is formed by a complex association of eumelaninand pheomelanin.

Their biosyntheses are common up to dopaquinone (double oxidation oftyrosine by tyrosinase, a cupro-proteinic enzyme). Their syntheticroutes then diverge.

Brown eumelanin is an indole-5,6-quinone polymer, whereas pheomelanin,which is responsible for the red color, is a compound containing closeto 10% sulfur and has a structure which is a polymer of cysteinyldopa.

Enzymes other than tyrosinase participate in the creation and control ofmelanins:

dopachrome oxidoreductase: which converts dopachrome into5,6-dihydroxyindole and controls melanogenesis in the absence oftyrosinase,

α-glutamyl transpepsidase converts glutathionedopa into cysteinyldopa,

the glutathione system (reductase-peroxidase) which controls thecrossroad between the biosynthesis of eumelanin and pheomelanin.

It appears that the surrounding sulfur level is the determining factorof such an orientation.

The glutathione reductase activity and the level of reduced glutathioneare higher in people with ginger pigmentation than in those with brownpigmentation.

Dopachrome tautomerase regulates the reaction of dopachrome5,6-dihydroxyindole-2-carboxylic acid and controls the proportion ofcarboxylated subunits in the melanin polymer.

Agents which depigment or whiten the complexion are chemical compoundsthat are capable of acting at the tissue, cellular or subcellular level.

They act on melanin itself or on the existence of melanocytes(melanocytotoxicity).

The general modes of action may be as follows:

inhibition of the formation of melanosomes,

adverse change in the structure of melanosomes,

inhibition of tyrosinase biosynthesis,

inhibition of melanin biosynthesis,

interference of the transfer of melanosomes into the keratinocytes,

chemical effect on melanin with an increase in the degradation ofmelanosomes in the keratinocytes.

Moreover, it is necessary to demonstrate and eliminate the factor whichinduces hyperpigmentation before treating it (U.V., perfume,oestroprogestative, and to recommend a maximum-type sun protectionthroughout the medical treatment).

The motivations which compel people to bleach their skin may be verydiverse.

Direct lightening of the skin is desired in Black Africa, withtraditional or chemical solutions which have considerable harmful sideeffects on the appearance and structure of the skin.

The pallor or whiteness of asiatic facial skin is obtained withmolecules which act with the least possible toxicity (arbutin, kojicacid and ascorbic acid).

The treatment of hyperpigmentation marks in white people requires theuse of diverse molecules, the main one of which, hydroquinone, is thesubject of intense scrutiny and its maximum cosmetic dose is 2%.

There is thus a need for compositions which have pronounced depigmentingactivity at moderate concentrations, and which are well tolerated by theskin.

Accordingly, the subject of the present invention is a dermatologicaland/or cosmetic composition, characterized in that it contains adepigmenting active extract obtained from mouse-ear hawkweed.

Mouse-ear hawkweed, Hieracium pilosella, belongs to the family ofCompositae. It is a small herbaceous ground-covering plant 10 to 30 cmin height.

The leaves form a basal rosette, they are long, oval and whitishunderneath and covered with a silky down on both sides.

The flower-bearing stem is upright, single and bears a capitulum withhermaphroditic flowers, all ligulated, pentadentate and pale yellow incolor. There are many bracts, often carrying black glandular hairs.

The fruit is a finely ribbed achene with aigrettes.

Mouse-ear hawkweed flowers from May to September.

It is found in dry locations, throughout almost all of Europe, in NorthAfrica and in North America. It is very common in France but rare in theMediterranean region.

Many chemical studies carried out on this plant have allowed a largenumber of molecules to be isolated and identified. Mouse-ear hawkweed ischaracterized by the presence of oxicoumarins: umbelliferone(7-hydroxycoumarin) and umbelliferone-7-monoglucoside or skimmine. Theumbelliferone content varies according to the organs, with the leavesbeing the richest, and according to the seasons; the content is maximumin summer and minimum at the end of winter.

Phenolic acids have also been identified, as well as flavonoids:apigenin and its derivatives, luteolin and its derivatives. Certainauthors note the presence of tannins throughout the plant.

Mouse-ear hawkweed has been used for its diuretic properties intraditional medicine. Its choleretic and cholagogic properties have alsobeen described: thus, FR 2,549,373 uses essence of mouse-ear hawkweedfor the preparation of a composition which aids digestion and bilesecretion. Patent FR 744 M has proposed it as a hypocholesterolemiant.Aqueous compositions containing silanol and extracts of mouse-earhawkweed have been proposed in FR 2,620,029 for the surface treatment oflymphatic vessels.

The Applicant has now revealed, unexpectedly, that active principleswhich can be prepared from mouse-ear hawkweed have properties that canadvantageously be exploited in dermocosmetic compositions; the Applicanthas revealed the depigmenting activity of an extract of mouse-earhawkweed.

An active extract may be obtained in particular by a process whichcomprises a step of extraction from the aerial parts and/or roots ofmouse-ear hawkweed, by a polar solvent chosen from the group comprisingwater, alcohol, acetone and mixtures thereof; in these aqueous-alcoholicand/or aqueous-acetone solvents, the proportion of each of theconstituents may range between 0 and 100.

According to one of the embodiments of the invention, the plant isground and then extracted with a C₁ to C₄ alcohol or with a water/C₁ toC₄ alcohol mixture or with acetone or with a water/acetone mixture, inwater/C₁ to C₄ alcohol or water/acetone proportions ranging from 100/0to 0/100. The plant/solvent ratio ranges from 1/5 to 1/20.

Preferably, the entire plant is ground and then extracted with a C₁ toC₄ alcohol or with a water/C₁ to C₄ alcohol mixture, in proportionsranging from 90/10 to 10/90.

The extraction may be carried out statically or with stirring, attemperatures ranging from the boiling point to room temperature. Theduration of the extraction is between 1 hour and 24 hours. Afterextraction, the solutions are recovered by filtration or draining.

It is possible, in a second stage, to carry out steps of enriching inactive principles.

The alcohols or the acetone are then evaporated off under vacuum, attemperatures between 40° C. and 100° C. The concentrated aqueoussolutions are purified by liquid/liquid extraction.

The organic solvents used are alkane-type solvents, such as hexane orheptane, chlorinated solvents such as dichloromethane and chloroform,butanol, ethyl acetate or solvents whose miscibility with water dependson the ionic strength, such as acetone, isopropanol and ethanol. In thiscase, the aqueous solution will be saturated with (NH₄)₂ SO₄, NaCl orNa₂ SO₄. The pH of the liquid/liquid extraction may be adjusted tobetween pH 2.5 and pH 8 depending on the extraction solvents.

The organic phases are recovered and filtered.

The extract may be used in the present state. The organic solvent mayalso be evaporated off under vacuum, at a temperature between 40° C. andthe boiling point of the solvent. The dry extract recovered is thenground in the form of a powder.

The various mouse-ear hawkweed extracts are assayed for theirumbelliferone content, by high performance liquid chromatography. Theassay is carried out on a C₁₈ column with a mobile phase: 95/5/15/0.5water/propanol/tetrahydrofuran/phosphoric acid relative to a purecontrol sample of umbelliferone.

The contents vary according to the degree of purification of theextract. Thus, the proportions of umbelliferone relative to the solidscontent of an unpurified extract range from 0.5 to 2%, and for apurified extract from 4 to 10%.

The invention also comprises the use of a product with a compositionsimilar to the mouse-ear hawkweed extract, but which may be prepared bysynthesis.

Preferably, the mouse-ear hawkweed extract is present with anumbelliferone titer of between 0.05% and 10% by weight of the totalcomposition.

The compositions according to the invention may be in the form oflotions, emulsions, creams, ointments, salves, etc. They also containany formulation excipient known to those skilled in the art which issuitable for good topical application. They especially containstabilizers, preserving agents, surfactants, fragrances and dyes.

In the compositions according to the invention, the active extract ofmouse-ear hawkweed may be combined with keratolytic compounds, forexample salicylic acid, lactic acid, glycolic acid or malic acid, aswell as with other α-hydroxy acids known to those skilled in the art.

According to another aspect of the invention, the dermocosmeticcompositions described above also contain a screening agent or asunscreen; such inorganic and/or organic screening agents are known tothose skilled in the art, who will adapt their choice and concentrationsdepending on the degree of protection desired.

The subject of the invention is also a cosmetic treatment method,characterized in that an extract of mouse-ear hawkweed is appliedlocally.

More particularly, the subject of the invention is the use, in acosmetic treatment method, of an active extract of mouse-ear hawkweed inorder to reduce and/or eliminate pigmentation marks.

Pigmentations often appear under the effects of UVA and B radiation,during prolonged exposure to sunlight.

The Applicant has thus evaluated the UV-screening andfree-radical-trapping activities of extracts of mouse-ear hawkweed.These activities contribute towards the depigmenting activity of theextracts of mouse-ear hawkweed.

One of the aspects of the invention thus concerns the use, in a cosmeticmethod, of an extract of mouse-ear hawkweed as an anti-UVA and anti-UVBscreening agent. It also relates to the use of an extract of mouse-earhawkweed as an anti-radical substance.

The depigmenting properties of extracts of mouse-ear hawkweed or of anactive product which may be prepared from mouse-ear hawkweed may,according to another aspect of the invention, lead to the use of anactive product which can be obtained from mouse-ear hawkweed for thepreparation of a medicinal product that is active as a depigmentingagent.

The pharmaceutical compositions will contain the extract of mouse-earhawkweed combined with pharmaceutically acceptable excipients.

The examples which follow are intended to illustrate the inventionwithout in any way limiting the scope thereof.

Reference will be made to FIG. 1 which represents the UV activity of anextract of mouse-ear hawkweed.

EXAMPLE 1

100 kg of whole mouse-ear hawkweed plants are ground and then extractedwith 800 kg of 20% ethanol with stirring at reflux for 1 hour. Theaqueous-alcoholic solution is recovered by draining and then filtered.It is then concentrated under vacuum at 60° C. until 100 kg of aqueoussolution are obtained.

The pH of this solution is brought to pH 3.5 by addition of hydrochloricacid.

A liquid/liquid extraction is carried out with 300 kg of ethyl acetatewith stirring for 2 hours. After separation of the phases by settling,the organic phase is recovered, concentrated under vacuum at 40° C. andthen dried and ground. 1 kg of dry extract is recovered, theumbelliferone content of which is between 6 and 8%.

EXAMPLE 2

1 kg of whole mouse-ear hawkweed plants is extracted with 5 kg of cold60% ethanol, for 24 hours. The aqueous-alcoholic solution is recoveredby filtration. Its umbelliferone content is between 1 and 2% relative tothe solids content.

EXAMPLE 3

10 kg of whole mouse-ear hawkweed plants are ground using a hammer mill.The plant meal is then extracted at reflux, with stirring, with 100 kgof a 50/50 methanol/water mixture. The solution is recovered by drainingand then filtered. It is then concentrated under vacuum at 50° C. until10 kg of aqueous concentrate are obtained. Ammonium sulfate is added tothis concentrate to saturation, followed by 30 kg of isopropanol.

The mixture is stirred for 2 hours and is then left to separate bysettling. The isopropyl phase is recovered, concentrated and then driedunder vacuum.

4.5 kg of a powder are obtained, the umbelliferone content of whichranges from 4 to 6%.

EXAMPLE 4

1 kg of whole mouse-ear hawkweed plants is ground and then extractedwith an 80/20 water/acetone mixture at reflux for one hour.

The aqueous-acetone solution obtained after filtration is concentratedand then dried under reduced pressure at a temperature between 40° and50° C. The dry extract obtained is ground and then titrated to between 5and 8% umbelliferone.

EXAMPLE 5

Evaluation of the sunscreen activity

The UVA and UVB sunscreen activity of the extract of mouse-ear hawkweedis evaluated by spectrophotometric study of the extract titrated to 5%umbelliferone.

This extract has an absorption maximum at 325 nm and a specificextension coefficient of 570 at 310 nm.

This demnonstrates the UVB screening potential of the extract ofmouse-ear hawkweed. By comparison, Parsol MCX, a chemical UVB screeningagent, has a specific extension coefficient of 810 at 310 nm.

The results are represented in the figure appended to the application.

EXAMPLE 6

Evaluation of the anti-radical activity

Radical reactions are divided into 3 successive steps: initiation,propagation and disappearance of the free radicals:

initiation is due to the superoxide anion O₂ ⁻ which appears under theeffect of factors such as UV radiation or stress,

propagation is brought about by the hydroxyl radicals OH,

disappearance of the free radicals. Free radical traps act on thesuperoxide anion O₂ ⁻ but also on the hydroxyl radicals. Thus, we havesought to evaluate the anti-radical activity on these two steps.

The activity on the superoxide anion is carried out by testing in vitro.The superoxide anion is generated by radical photo-oxidation, bysensitization of riboflavin with visible radiation.

The colored indicator used is tetrazolium nitro blue, an electrophile,which is reduced by the superoxide anion generated to diformazan.

The anti-radical activity of the extract of mouse-ear hawkweed isexpressed by the concentration of extract which inhibits 50% of thereductive activity of the superoxide anion on TNB, the IC₅₀. For anextract of mouse-ear hawkweed titrated to 5% umbelliferone, the IC₅₀ is0.3 mg/ml.

The activity on radical propagation, and thus on the hydroxyl radicals,is evaluated on DPHH, diphenyl-picrylhydrazyl hydrate, which is astable, colored free radical. The IC₅₀ of an extract of mouse-earhawkweed containing 5% umbelliferone is 0.025 mg/ml. In this test,vitamin E has an IC₅₀ of 0.006 mg/ml.

EXAMPLE 7

Evaluation of the depigmenting activity in vitro

In vitro, an inhibitory activity on the activity of tyrosinase, the mainenzyme involved in the pigmentation process, is desired. In the presenceof oxygen, the reaction of tyrosinase with tyrosine is reflected by anincrease in the optical density of the reaction medium at 280 nm.

Any inhibition of the action of tyrosinase on tyrosine, directly on theenzyme or indirectly by competition with tyrosine, results in a smallerincrease in the optical density at 280 nm.

The IC₅₀, the concentration of extract of mouse-ear hawkweed whichinhibits the tyrosinase reaction signal by 50%, is calculated.

For an extract of mouse-ear hawkweed assayed at 5% umbelliferone, theIC₅₀ is 30 μg/ml.

EXAMPLE 8

Depigmentation in vivo

Evaluation of the depigmenting activity consists in searching for aninhibitory effect of topical preparations containing an extract ofmouse-ear hawkweed titrated to 5% umbelliferone on the hyperpigmentationof the caudal epidermis, induced by exposure of pigmented mice toultraviolet radiation.

The topical preparation is applied 7 days a week for 6 consecutiveweeks, with UV irradiation 5 days a week for 42 days.

The animals are subjected to a daily examination in order to monitor thechanges in pigmentation and to evaluate their intensity. The colorationsare assigned values according to a color scale.

The animals treated with the extract of mouse-ear hawkweed exhibitnon-uniform depigmentation when compared with the irradiated controlbatch. This effect cannot be attributed to a UV screening effect sincethe products are applied after the irradiation.

After the 42 days of treatment, the skin of the caudal appendix of theanimals is removed after they have been sacrificed. The optical densityat 700 nm is measured on the sample of epidermis.

A significant decrease in the optical density of the epidermis of theanimals treated with extract of mouse-ear hawkweed is observed. Thisdecrease reflects the depigmenting effect of this extract.

Lastly, the distribution and quantification of melanin in the layers ofthe epidermis are evaluated by image analysis.

A 24% overall decrease in melanin is observed, which is found both inthe basal and in the surface layers of the epidermis of mice treatedwith the extract of mouse-ear hawkweed.

EXAMPLE 9

Depigmenting formulae

The following formulae may be given by way of example:

    ______________________________________    A - Depigmenting lotion    ______________________________________    Extract of mouse-ear hawkweed with a 4% titer                            10 g    Propylene glycol        20 g    Ethyl alcohol           10 g    Distilled water qs      100 g    ______________________________________

The extract of mouse-ear hawkweed is dissolved in the propyleneglycol/ethyl alcohol mixture and then completed with distilled water.

    ______________________________________    B - Aqueous gel    ______________________________________    Extract of mouse-ear hawkweed with an 8% titer                              5 g    Propylene glycol         20 g    Ethyl alcohol            15 g    Hydroxypropylcellulose   1.5 g    Distilled water qs       100 g    ______________________________________

The extract of mouse-ear hawkweed is dissolved in the propyleneglycol/ethyl alcohol mixture and then completed with distilled water.

The cellulose is introduced with stirring until the gel forms.

    ______________________________________    C - Emulsion    ______________________________________    Extract of mouse-ear hawkweed with a 4% titer                            3 g    Glucose ester (Glucate SS)                            4 g    Ethoxylated glucose ester (Glucate SSE 20)                            4 g    Thick liquid petroleum jelly                            10 g    C.sub.8 -C.sub.10 triglycerides                            4 g    Cyclomethicone (Dow Corning 345)                            4 g    Carboxyvinyl polymer (Carbomer 940)                            1 g    EDTA, 2 Na              0.2 g    Distilled water qs      100 g    ______________________________________

EXAMPLE 10

Roll-on containing depigmenting fluid

    ______________________________________    Extract of mouse-ear hawkweed                       0.1 to 10%    95° alcohol 35 to 75%    Isopropyl adipate   5 to 15%    Oleic acid         0.01 to 1%    Propylene glycol   10 to 40%    Klucel MF          0.1 to 2%    Water qs           100    ______________________________________

EXAMPLE 11

Depigmenting/anti-radical/keratolytic fluid

    ______________________________________    Extract of mouse-ear hawkweed                       0.1 to 10%    Salicylic acid     0.1 to 5%    Vitamin C PCA      0.1 to 10%    Hydroquinone       0.1 to 5%    Batyl alcohol      0.05 to 1%    95° alcohol 10 to 75%    Isopropyl adipate   5 to 15%    Oleic acid         0.01 to 1%    Propylene glycol    5 to 40%    Klucel MF          0.1 to 2%    Water qs           100    ______________________________________

EXAMPLE 12

Depigmenting day cream

    ______________________________________    Arlacel 165 (ICI)   5 to 15%    Cetyl alcohol      0.1 to 3%    Stearic acid       1 to 6%    Liquid paraffin     2 to 15%    Isopropyl isostearate                       1 to 6%    Sunflower oil      1 to 2%    Extract of mouse-ear hawkweed                       0.1 to 25%    Batyl alcohol      0.05 to 1%    AHA                0.1 to 25%    Propylene glycol   0.1 to 10%    Water qs           100    ______________________________________

EXAMPLE 13

O/W depigmenting roll-on

    ______________________________________    Arlamol E          0.1 to 10%    Brij 72            1 to 3%    Brij 721           1 to 5%    Extract of mouse-ear hawkweed                       0.1 to 25%    Oleic acid         0.05 to 1%    Water qs           100    Alcohol             0 to 10%    ______________________________________

EXAMPLE 14

O/W depigmenting milk

    ______________________________________    Arlatone 985       1 to 5%    Brij 721           1 to 3%    Miglyol 812         1 to 10%    Arlamol MD          1 to 10%    Extract of mouse-ear hawkweed                       0.1 to 25%    Atlas G 2330       0.1 to 5%    Salicylic acid     0.1 to 2%    Alcohol             0 to 10%    α-Bisabolol  0.05 to 0.5%    Magnesium vitamin C phosphate                       0.1 to 3%    Water qs           100    ______________________________________

EXAMPLE 15

Moisturizing and depigmenting emulsion protective against UVB and A

    ______________________________________    Extract of mouse-ear hawkweed                       0.1 to 25%    Arlacel 2121        1 to 10%    Arlamol HD          1 to 10%    Alcohol             0 to 10%    Oleic acid         0.1 to 1%    Behenyl alcohol    0.1 to 2%    Tocopheryl acetate 0.05 to 1%    Glycerol           0.01 to 10%    AHA                0.1 to 25%    Vitamin C PCA      0.1 to 3%    Glycerol           0.1 to 15%    TIO.sub.2           0 to 25%    ZnO                 0 to 25%    Cinnamate           0 to 10%    Dibenzoylmethane   0 to 4%    Water qs           100    ______________________________________

EXAMPLE 16

Depigmenting W/O emulsion night cream

    ______________________________________    Arlacel 481         2 to 10%    Paraffin            1 to 20%    Glycerol            1 to 15%    MgSO.sub.4         0.5    Water qs           100    Extract of mouse-ear hawkweed                       0.1 to 25%    ______________________________________

EXAMPLE 17

Nourishing depigmenting emulsion

    ______________________________________    Arlacel 1689       1 to 5%    Miglyol 812         1 to 15%    Aerosil 972        0.1 to 0.5%    Glycerol            1 to 15%    MgSO.sub.4         0.1 to 0.5%    Water qs           100    Extract of mouse-ear hawkweed                       0.1 to 25%    Batyl alcohol      0.05 to 0.3%    ______________________________________

EXAMPLE 18

Depigmenting stick

    ______________________________________    Super Hartolan       9%    Lunacera Alba      4.5%    Lunacera C 40      7.2%    Lunacera C 46      3.7%    Lunacera M         4.5%    Petroleum jelly     0 to 18%    Castor oil          0 to 28%    Isopropyl myristate                        0 to 30%    TIO.sub.2 - Mica (Timica Silk blue)                       0.1 to 5%    Ultra-fine TIO.sub.2                        0 to 25%    Ultra-fine ZnO      0 to 25%    Extract of mouse-ear hawkweed                       0.1 to 25%    Batyl alcohol      0.05 to 1%    Carotene           0.005 to 0.1%    Abil WE 09         0.1 to 2%    Water qs           0 to 5%    ______________________________________

EXAMPLE 19

Depigmenting ultra-fine spray-emulsion

    ______________________________________    Ethoxylated behenyl alcohol (Mergital B 10)                            5 to 10%    Vegetable oil          0.1 to 5%    Lanette 22             0.1 to 2%    Extract of mouse-ear hawkweed                           0.1 to 25%    Vitamin C palmitate    0.1 to 3%    AHA                    0.1 to 25%    Bisabolol              0.05 to 0.5%    Water qs               100    ______________________________________

We claim:
 1. A method for reducing or eliminating excessive pigmentationmarks comprising locally administering to a subject in need of suchtreatment an effective amount of an extract of mouse-ear hawkweed.
 2. Amethod according to claim 1, characterized in that the extract isobtained after a step of extraction by a solvent chosen from the groupconsisting of water, alcohol, acetone and mixtures thereof in allproportions, from the aerial parts and/or the roots of mouse-earhawkweed.
 3. A method according to claim 2, characterized in that theextract is obtained after a step of aqueous-alcoholic extraction, with awater/alcohol ratio of between 90/10 and 10/90, from the aerial partsand/or the roots of mouse-ear hawkweed.
 4. A method according to claim1, comprising coadministering at least one other depigmenting activeprinciple.
 5. A method according to claim 1, comprising coadministeringat least one organic or inorganic sunscreen.
 6. A method according toclaim 1, comprising coadministering at least one keratolytic activeprinciple.
 7. A method according to claim 1, characterized in that theextract of mouse-ear hawkweed is present in an umbelliferone titer ofbetween 0.05% and 10% of the total composition.
 8. A method according toclaim 1, in which the extract of mouse-ear hawkweed also acts as ananti-UVA and B screening agent.
 9. A method according to claim 8, inwhich the extract of mouse-ear hawkweed also acts as an anti-radicalsubstance.